NL8204925A - IMPROVEMENTS IN AND RELATING TO A DEVICE FOR DEPRETING VEGETABLE REMANANCE, IN PARTICULAR Soybean MENTION, A PROCESS FOR THE PREPARATION OF A PURIFIED VEGETABLE PROTEIN PRODUCT AND THE PURIFIED VEGETABLES. - Google Patents

Description

• - V. 1 / Improvements In and with respect to a means for decomposing vegetable remanence, in particular soy mane, a process for preparing a purified vegetable protein product as well as the purified vegetable protein product.

A process for the preparation of a purified vegetable protein product (PVP) by enzymatic removal of the remanence, without dissolution and reprecipitation of the protein is described in Belgian patent 882,769. Also described in this patent is the importance of keeping the proteolytic activity as low as possible. The purity of the pvp obtainable by the known method is not satisfactory and can therefore be improved. In the examples, a purity of the PVP of about 85% was demonstrated. Even if it is possible to obtain a pvp 10 with a purity of about 90% by the known method, it is only available with certain pre-treated starting products, for example soy protein concentrate. It would be desirable to be able to obtain a pvp purity of greater than 90% with a much wider spectrum of starting materials, in particular husked and degreased soybean meal.

The invention is based on the surprising finding that a certain portion of the remanence decomposition product, as it occurs during the above enzymatic treatment, ie, a high molecular weight water-soluble carbohydrate, itself attaches to a portion of the protein, as detailed later will be explained. This, of course, results in a lower purity of the protein.

Therefore, it is an object of the present invention to provide a means for decomposing vegetable retentivity in the presence of vegetable protein, wherein the vegetable retentivity is in particular soy manhood, which will result in an improved purity pvp and process to prepare a pvp.

The basis for the invention can be described in the following manner, referring to Fig. 1, wherein only products consisting of as undissolved solids are indicated, with all of the above layers omitted. An amount of soybean meal was divided into two equal parts, Part I and Part II (column a in Fig. 1).

Part I was proteolytically decomposed at a pH of about 8 by ALCALASE® (a proteolytic enzyme prepared by 35 µl. Lchenchenis and marketed by NOVO INDUSTRI A / S, 2880 8204925 * * 2

Bagsvaerd, Denmark) and then further washed at about pH 8 to eliminate the total amount of protein and the remanence was separated from the supernatant and washed (see part I, columns a and b, fig. 1). In this way, a pure remanence (denoted -5 denoted by remanence I) was isolated (column b in Figure 1). Part II of the soybean meal was not treated; for brevity, the remanence in part II is referred to as remanence II (column b in fig. 1). Now both retentivity I and part II are decomposed by means of a commercial pectinase, for example PECTINEX® (a pectolytic enzyme prepared by Schweizerische Ferment AG, Basel, Switzerland) (see columns b and c in Fig. 1). Surprisingly, it was found that the undissolved part of retentivity I is much smaller than the undissolved part of retentivity II based on nitrogen and dry matter mass balance, see Fig. 1, where the shaded areas in column c correspond to the insoluble, non-protein products in the above step. Furthermore, when the supernatant from the pectinase-treated remanence I is brought to pH 4.5 together with a soy protein suspension, a polysaccharide in the supernatant layer is bound to the soy protein. This polysaccharide in the above layer of remanence I, which is a part of the remanence decomposition product and which is clearly soluble in water in the absence of soy protein, but bound to soy protein at or about the isoelectric point of soy protein, when soy protein is present, is denoted SPS (soluble polysaccharide), see Fig. 1. The SPS has a molecular weight distribution between 5 x 10 ^ and 4.9 x 10 ^. The preparation of isolated SPS

appears from the flow chart given in Figure 2, which also includes some of the processes presented in Figure 1. Therefore, the problem is to find an agent capable of decomposing the SPS in such a way that the SPS decomposition products do not bind soy protein or bind soy protein to a much lesser degree than SPS binds soy protein.

Although the description mainly refers to the decomposition of soy protein, the invention is not limited to soy protein, but includes all types of vegetable proteins, see for example the proteins listed in Belgian patent 882,769 on page 1.

It has now been found according to the invention that by comparative research on the ability to decompose soybean SPS, it is possible to select mirco-organisms capable of producing a compound exhibiting an enzymatic activity, which effectively soy 40 SPS decomposed, referred to in the following for brevity as SPS-ase.

8204925% 3 <Accordingly, it can be stated that an SPSase is a carbohydrase capable of decomposing soybean SPS under suitable conditions to decomposition products which themselves adhere to vegetable protein in an aqueous medium to a lesser degree than the soybean SPS 5 prior to decomposition would attach itself to the same vegetable protein under similar conditions.

Therefore, in a first aspect, the invention includes an agent for decomposition of vegetable remanence, in particular soy maneness, in the presence of vegetable protein, in particular soy protein-10in, suitable for the preparation of a pvp with a protein purity of about 90% with a vegetable protein, which may be degreased or partially degreased, as a starting product, containing an enzyme with remanence solubilizing activity, the agent containing an enzyme capable of decomposing soy SPS (SPS-ase) and the agent is substantially free of proteolytic activity. The agent is substantially free of proteolytic activity when the proteolytic activity is equal to or less than the proteolytic activity, which will be accompanied by a total protein loss in the finished pvp of no more than 30%, preferably no more than 15% , more preferably not more than 10%, when about 65% of the remanence is solubilized, based on a nitrogen and dry matter mass balance ·

It has been found that this SPSase, which can degrade soybean SPS, is capable of degrading polysaccharides in a manner similar to SPS and fruit and vegetable origin, in a more complete manner than commercially available pectinases and in commercially available cellulases.

By total or partial removal of the SPS from the final vegetable protein, the purity of the final vegetable protein is necessarily improved compared to the purity of the final vegetable protein, which is available according to the method known from Belgian Patent 882,769, as this known vegetable protein product contaminated with SPS.

It is currently not known whether the particular SPS-ase described below has its enzymatic activity of a single enzyme or of an enzyme complex containing at least two enzymes. Some studies seem to indicate that at least two enzymes are responsible for the SPSase degradation effect, with one of these enzymes capable of only minor decomposition of SPS, 40 after which one or more enzymes are capable of a more extensive execute breakdown 8204925 * *% 4 of the SPS. However, applicants do not wish to be limited by such a hypothesis or similar hypotheses.

A preferred embodiment of the agent according to the invention is characterized by the fact that the SPSase was produced by means of a micro-organism belonging to the genus Aspergillus.

A preferred embodiment of the agent according to the invention is characterized in that the active component comes from the enzymes which can be produced by means of Asp. aculeatus CBS 101.43. The same SPSase can be generated by Asp, dress IFO 4408. It was found that Asp. aculeatus CBS 101.43 also produces very potent remanence solubilizing enzymes, cellulases, pectinases and hemicellulases. Furthermore, it was found that not every and every strain belong to the species Asp. aculeatus or Asp. japonicus to develop an SPS axis necessary for the invention. Therefore, as will be apparent from a later section in this specification, it has been shown that the Asp. japonicus ATCC 20236 does not generate such amounts of an SPS-ase that can be determined by the enzymatic determination of SPS-ase described herein.

A preferred embodiment of the agent according to the invention is characterized in that the SPSase is immunoelectrophoretic identical to the SPSase which can be produced by Asp. aculeatus CBS 101.43 and identifiable by the immino electrophoretic coating technique, see sections 6 and 7.

A preferred embodiment of the agent according to the invention is characterized in that the ratio between the proteolytic activity in HUT units and the remanence solubilizing activity in SRUM-120 units is less than about 2: 1, preferably less more than 1: 1, more preferably less than 0.25: 1. It was found that there is a pure correlation between the protease activity expressed in HUT units (to be defined later) at pH 3.2 (the pH activity optimum of the protease) and the protein loss. This correlation is specific for the SPSase preparation to be prepared by Asp. aculeatus CBS 101.43. It should be understood that this correlation 35 cannot exist in relation to another SPS-axis-forming microorganism that forms an SPS-axis different from the SPS-axis, which can be generated by CBS 101.43 however, one skilled in the art will be able to find a similar correlation that will fulfill the protein loss requirement set forth in the first claim. The SRUM-120 activity (to be defined later) is a measure of 8204925 * 5% the usual remanence solubilizing, cellulase, pectinase and hemicellulase actives and other activities. The SRUM-120 units are measured at a pH of 4.5. It should be understood that this pH is selected because the decomposition of the retentivity is performed at or approximately at the isoelectric pH of the soy protein and that a different enzyme activity method should be used if the decomposition of the retentivity is performed at another pH value.

A preferred embodiment of the agent according to the invention is characterized in that the agent contains cellulase activity and that the cellulase activity originates in part or in whole from Trichoderma reseel. This cellulase is able to dissolve crystalline cellulose and the agent gives rise to a high purity PVP.

A preferred embodiment of the agent according to the invention is characterized in that the agent contains cellulase activity (Οχ), pectinase activity (Pü, PGE, UPTE, PEE) and hemicellulase activity (VHCU).

In Agr.Biol.Chem 40 (1), 87-92, 1976, it is described that a strain of Asp, japonicus ATCC 20236, produces an enzyme complex capable of partial decomposition of an acidic polysaccharide in soy sauce, called APS , a fraction of which is indicated to carry APS-I. This acidic polysaccharide is not identical to SPS, as will be shown in more detail in section 3 later in the description.

Therefore, the HPLC gel filtration chromatograms of SPS and APS are markedly different, and further, the gel filtration chromatograms of APS decomposed by the commercial spectinase Pectolyase and of SPS treated with the commercial spectinase Pectolyase are distinctly different. Furthermore, it does not appear from the article that the acidic polysaccharide is bound to the soy protein and that the decomposed acidic polysaccharide is not bound to the soy protein or to the soy protein to a much lesser degree than the undecomposed acidic polysaccharide.

It has also been shown that this strain does not form an SPSase in such amounts as can be determined by the enzymatic determination of SPSase as described herein. This creates a front ear portion against each strain of Asp. japonicus as a production agent of an SPSase, but surprisingly it has been found according to the invention that some strains of Asp. japonicus generators are from an SPS axis.

The invention, in a second aspect, comprises a method of preparing a purified vegetable protein product by removing remanence from an impure vegetable protein serving as starting material, the starting product being treated with the agent according to the invention. invention in an aqueous medium at a pH value which differs by no more than 1.5 pH units from the isoelectric point 5 of the main portion of the protein portion of the starting product and at a temperature between about 20 and about 70 ° C until at least about 60% of the remanence, based on the nitrogen and dry matter mass balance, preferably at least about 70% thereof, more preferably at least about 80% thereof, is solubilized, followed by separation of the solid phase containing the purified vegetable protein product from the above layer.

A preferred embodiment of the method according to the invention is characterized in that the separation is carried out at a temperature between room temperature and the freezing point of the above layer. Hereby a high protein yield is obtained.

A preferred embodiment of the method according to the invention is characterized in that the starting product is degreased or degreased and furthermore partly purified vegetable protein. This output product is readily available.

A preferred embodiment of the method according to the invention is characterized in that the starting product is soybean meal. This starting product is good and easily available.

A preferred embodiment of the method according to the invention is characterized in that the starting material is heat-treated soybean meal, preferably with the jet of boiled soybean meal. A lower enzyme dosage can be used here and a high efficiency can furthermore be obtained.

A preferred embodiment of the method according to the invention is characterized by the fact that the starting product is able to pass a sieve with a mesh size of about 2.5 nm. This ensures a reasonably short response period.

Only the dosing rate (in SAE units) for the SPS-ase activity for the treatment of soybean meal can be provided, with at least about 35 SAE units per 100g jet of cooked soybean meal and 350 SAE units per 100g of uncooked soybean meal. As a practical matter, the proper relationship of the enzyme activity required for SPS degradation is not known and therefore minimum dosages and ratios for pectinase, cellulase and hemicellulases 40 cannot be provided. However, a compilation activity for the 8204925 ». Treatment of soybean meal In SRUM units, n.1. Provides at least about 60 SRUM-120 units per 100 g for boiled soybean meal, 600 for unheated meal. The example values provided below are believed to be more than The Minimum Dosage Trials Experiments are used to determine the optimum operating amounts and reaction times for the soy substrate to be converted to PVP.

The invention includes in a third aspect a purified vegetable protein product obtained by the method of the invention.

In order to illustrate the nature of the invention, reference is made to the following sections 1-10, which describe all the details associated with the invention: 1. Production of SPS.

2. Characterization of SPS, in particular its molecular weight distribution.

3. Documentation that SPS and APS are different connections.

4. Comparative study for SPSase-producing microorganisms.

20 5. Identification of some SPS-ase forming microorganisms.

6. General description of the coating technique associated with immunoelectrophoresis.

7. Immunoelectrophoretic labeling of SPSase with polyspecific antibody and coating.

25 8. Purification of an SPS-ase preparation.

9. pH activity dependence, temperature dependence and stability of an SPS axis.

10. Enzymatic activity determinations.

SECTION 1.

30 PREPARATION OF SPS.

As mentioned above, the starting material for the preparation of SPS can be soy manhood. Therefore, in the first place, the preparation of soy mania is described.

Soy manhood is the protein-free carbohydrate fraction (which may contain minor amounts of lignin and minerals in practice) in defatted and husked soybean meal, which carbohydrate fraction is insoluble in an aqueous medium at pH 4.5 and can be obtained in the following manner, also referring to to fig. 1.

Defatted soybean meal (Sojamei 13 from Aarhus Oliefabrik A / S) is suspended in 40 deionized water at 50 ° C in a weight ratio of soybean meal: water 8204925 8 = 1: 5 In a tank with pH condition and temperature control. The pH is adjusted with 4 N NaOH (I). Now a pH-state hydrolysis is carried out with ALCALASE 0.6 L (a proteolytic enzyme based on JJ. Licheniformis with an activity of 0.6 Anson units / g, the activity being determined according to the Anson method as described in NOVO ENZYME INFORMATION IB No. 058 e-GB), the enzyme / substrate ratio being equal to 4% of the amount of protein in the soybean meal (II). After a 1 hour hydrolysis, the sludge is separated by centrifugation (III) and washing (IV), this operation being performed twice (V, VI, VII). The sludge thus treated is hydrolysed once more for 1 hour with ALCALASE No. 0.6 L (VIII, IX) similar as indicated above. The sludge is then separated by centrifugation (X) and washed twice (XI, XII, XIII, XIV), the final washed sludge (VI) being subjected to a spray drying treatment (XV). The powder thus produced by spray drying is the soy mane, which serves as the raw material for the production of SPS.

SPS is the water-soluble polysaccharide fraction, which is formed by the usual treatment of the above-mentioned so-20-year-old with pectinase. The SPS is generated in the following manner by the 14 reaction steps indicated below, also referring to Figure 2.

1. The dry matter content in the above-mentioned soy manners is determined and the soy manners are diluted with water to 2% dry matter and kept in suspension in a tank under temperature control at 50 ° C.

2. The pH value is adjusted to 4.50 with 6 N NaOH.

3. Pectinex Super cone. L is added in an amount of 200 g / kg of dry matter (a commercial spectinase of Schweizerische Ferment 30 AG, Basel, Switzerland with a pectinase activity of 750,000 MOU, as determined in the brochure "Determination of the Pectinase units on Apple Juice (MOU ) ”Of 12.6.1981, available from Schweizerische Ferment AG, Basel, Switzerland) and also Celluclast 200 L is added in an amount of 200 g / kg dry matter, a commercial cellulose described in the brochure NOVO enzymes, information sheet B 153 e-GB 1000 July, 1981, available from NOVO INDUSTRI A / S, Novo Alle, 2880 Bagsvaerd, Denmark).

4. The contents of the tank are kept at 50 ° C with stirring for 24 hours.

40 5. The enzymes are activated by increasing the pH value with 4 N NaOH to 8204925 9! § • * '1% 9.0. The reaction mixture is kept for 30 minutes and the pH value is adjusted again to 4.5 with 6 N HCl.

6. The reaction mixture is centrifuged and both the centrifuge product and the sludge are collected.

7. The centrifuge product from step 6 is filtered under control on a filter press (the filter is washed with water before the control filtration).

8. The control filtrate is ultrafiltrated, diafiltrated and further ultrafiltrated on a membrane with a limit value of 30,000 (DOS GR 60-P by De Danske Sukkerfabrikker), using the following parameters: 1 «Ultrafiltration corresponding to a volume concentration of 6.

2. Diafiltration until the percentage of dry matter in the permeate is 0 (* νθβ Brix).

3. Ultrafiltration to about 15% dry matter in the concentrate.

The temperature is 50 ° C, the pH is 4.5 and the average pressure is 3 bar.

9. The ultra-filtered concentrate is cooled to 5 ° C and an equal volume of 96% ethanol is added.

10. The precipitate is collected by centrifuge.

11. The precipitate is washed twice with 50% by volume ethanol in water, corresponding to the volume of the centrifuge product from step 10, i.e. two centrifugations are performed.

12. The washed precipitate is redissolved in water with a volume equal to the volume of the ultra-filtered concentrate from step 9.

13. The liquid from stage 12 is filtered under control over a glass filter.

14. The transparent filtrate, which contains pure SPS, is lyophilized.

8204925 «10

Flow chart No. 1 Degreased soybean meal H2O NaOH to pH 8 J_ T 1

Hydrolysis mix el I_ _ * · -

Alcalase 0.6 L ^ Hydrolysis 1 h before release II (E / S “4%) pH state (pH = 8, T = 50 ° C) 4N NaOH_» _

Centrifugation III centrifuge product 1 sludge waste 1

HpO le was 1 IV

4 * _____ _____ —_______ 1 2nd centrifugation Centrifuge product 2 sludge waste 2

H90 ^ 2nd was VI

3rd centrifugation VII centrifuge product 3 sludge waste 3 H? 0 New hydrolysis mixture VIII NaOH until pH = 8 t

Alcalase 0.6 L ^ Hydrolysis 1 hour before release IX 4N NaOH_ pH state (pH 8.0, T 50 ° C

8204925

Flow Diagram No. 1 (Continued) '%% 11 4th Spin X Centrifuge Product 4 ^ Sludge Waste 4

It was IV

5th centrifugation XII centrifuge product 5 sludge waste 5

H 2 O 2nd was XIII

6th centrifugation XIV centrifuge product 6 ^ sludge waste 6 spray drying__XV_ 8204925

Flow chart No. 2 * 12

1000 g Pectlnex Super conc »L 5 kg spray dried remanentle 100 g Celluclast 250 S

__i_ £ _

Decomposition with pectase 247 1H 2 O 4 and cellulase 1, 2, 3, 4, 5 50aC; pH - 4.5; t B 24 hours_ _i_

Spin sludge} 6 38 kg

Control filtration '**' 'T

Ultrafiltration, diafiltration, 18 1 HpO ^ ultrafiltration 241 1 permeate ^ (GR 60P) (19 - 26 °) _ 8 _i_ 5 1 50% C? HsOH j Pre-precipitation _ top layer ^ 9 (waste) _ * _

Spin centrifuge product ^ 10 precipitate _ * _ 2 x Wash__2 x centrifuge product 11 precipitate 8204925

Flow Chart No. 2 (Continued) 13 ft.

2 1 H? 0 ^ Re-solve 12 4 /

Control fltratle protein sludge ^ 13 (waste) _x_

Lyoflllserlng

310 g of lyophilized SPS

8204925 14 SECTION 2.

CHARACTERISTICS OF SPS, IN PARTICULAR ITS MOLECULAR WEIGHT DISTRIBUTION.

Gel chromatography on HPLC equipment (Waters pump 5 model 6000, Waters data module 730, and Waters refractometer R 401) determines the molecular weight distribution of the SPS, the production of which is performed as indicated in this description (Fig. 4). By the same method, the molecular weight distribution of the decomposition products of SPS is also determined by means of SPSase (Fig. 5). Furthermore, by the same method, the binding effect between soy protein and SPS (Fig. 6) and the absence of binding effect between soy protein and SPS decomposed by the agent of the present invention (Fig. 7) is demonstrated.

The calibration curve (the logarithm of the molecular weight plotted against 15 Rf, where the Rf value for glucose is arbitrarily defined as 1 and the Rj value for a specific dextran is defined as the retention time for this dextran divided by the retention time for glucose) determined by some standard dextrans with known molecular weights (T 4, T 10, T 40, T 70, T 110, T 500) from 20 Pharmacia Fine Chemicals AB, Box 175, S-75104, Uppsala, Sweden. The Rf value for the maximum of each dextran peak is found and the corresponding molecular weight is calculated as ^ Mj, * Μη, where the average value is the molecular weight by weight and Mn is the average value of the molecular weight by number. 0.1 M NaNO 3 is used as an eluent for this chromatographic method. The columns used in the chromatographic method are 60 cm PW 5000 followed by 60 cm PW 3000 from Toyo Soda Manufacturing Co., Japan. In this way, the relationship between the molecular weight and the Rf for the aforementioned dex-30 tears is established, see Fig. 3.

From FIG. 4, it can be calculated that SPS has a molecular weight distribution giving rise to a value of about 5.4 x 10 ^ and a value of Mjj of about 4.2 x 10 ^. It also appears from this figure that the chromatogram 35 shows two different peaks at a retention time of 34.5 min. (6%) corresponding to a molecular weight of about 5 x 10 ^ and a retention time of 47.12 min. (67%) corresponding to a molecular weight of about 4.9 x 10 ^. It also appears from this curve that a shoulder exists between these two peaks at a retention time of 41.25 40 min. (27%) corresponding to a molecular weight of 2.8 × 10 4.

8204925 15 'After decomposition of SPS with SPS-ase, the hydrolysis mixture was filtered through a membrane and the filtrate was chromatographed. It was found that about 55% SPS is decomposed into mono-, dl and trl saccharides and the remaining 45% is decomposed into a three-peak polymer 5 having the following molecular weights: 5 x 10 ^, 10 ^ and 4, 4 x 1 (P, see fig. 5.

In order to demonstrate the binding effect between soy protein and SPS and the significant reduction of the binding effect between soy protein and SPS decomposed by means of an SPS ase, the following 10 experiments were performed.

3% SPS in 0.10 M acetate buffer at pH 4.5 is added to an isolated soy suspension (Purina E 500) to produce a suspension with an isolated product / SPS ratio of 10: 1. This suspension is incubated on a shaking bath at 50 ° C for 18 hours. After the incubation, the suspension is centrifuged and the transparent supernatant is analyzed by HPLC as described above. From Fig. 6 it can be seen from comparison with Fig. 4 that the SPS is completely adsorbed to the isolated soy product.

The same method as indicated in the preceding paragraph 20 is performed with a 3% SPS solution hydrolysed with an SPS-ase generated by CBS 101.43 (Fig. 7). A comparison between Fig. 7 and Fig. 5 shows that no compound is adsorbed into the isolated soy product in the hydrolyzed SPS having a molecular weight below about 10 µm. The hydrolysis reduces the quantitative binding to about 10-15% in relation to the binding of SPS to soy protein.

An NMR analysis of the SPS, the preparation of which has been carried out as indicated in the present description, gives the following approximate composition of the SPS: 1) α-galacturonic acid in an amount of about 45%, with about 40% of the total amount of orgalacturonic acid is present as the methyl ester, 2) rhamnopyranose in an amount of about 20%, 3) galactopyranose in an amount of about 15% and 4) 0-xylopyranose in an amount of about 20%.

The constituents appear to be in a structure consisting of a rhamnogalacturon backbone and xylose and galactose side chains.

Complete acid hydrolysis of SPS (8 hours in 1 N sulfuric acid) and 40 subsequent thin layer chromatographic analysis indicated that 8204925 16 contained only minor amounts of the monosaccharides glucose and arabinose in the hydrolyzed SPS.

An HPLC analysis of the SPS decomposed by the SPSase enzyme complex formed by CBS 101.43 shows an impressive reduction in molecular weight. Accordingly, the NMR spectrum of the SPS decomposed as indicated above shows that most of the ester groups have disappeared and that the xylose and galactose content in the higher molecular weight product has also decreased. The NMR spectrum of the part of the SPS decomposition product which precipitates by adding 1 part by volume ethanol to 1 part by volume SPS decomposition product is similar to the NMR spectrum of the SPS, with the modifications indicated above, with with regard to the ester groups and the content of xylose and galactose.

SECTION 3 15 DOCUMENTATION ON THE FACT THAT SPS AND APS ARE DIFFERENT CONNECTIONS.

APS was prepared as indicated in Agr. Biol. Chem., 36, No. 4, 544-550 (1972).

Now this polysaccharide and SPS were hydrolyzed with various enzymes, after which the decomposition mixture was gel chromatographed on HPLC equipment, as indicated in section 2, "Characterization of SPS, in particular its molecular weight distribution".

In more detail, the hydrolyses were performed by treating 25 ml of solution of either 2% APS or 2% SPS in 0.1 M acetate buffer of pH 4.5 with 10 mg of KRF 68 or 30 mg of Pectolyase. KRF 68 is an SPS-ase preparation, the preparation of which is described in Example I. The results are shown in the following table.

8204925 17

Poly- HPLC gel Polysaccharide saccharide Enzyme chromatogram not decomposed 5_____ APS Pectolyase fig. 8 x APS KRF 68 fig. 9 x 10 SPS Pectolyase fig. 10 x SPS KRF 68 fig. 5 x 15 SECTION 4 COMPARISON ON SPS-ASE PRODUCING MICROORGANISMS .

The microorganism to be examined is incubated on an inclined agar substrate with a composition that allows the growth of the microorganism. After initial growth on the sloped agar substrate 20, the microorganism is transferred to a main liquid substrate, wherein the main carbon source is SPS (prepared as indicated), wherein the nitrogen source is ΝΟβ ”, ΝΗ ^ *, urea, free amino acids, proteins or other nitrogen containing compounds , which further contains a mixture of necessary salts and vitamins, preferably in the form of yeast extract. The composition of the main substrate depends on the micro-organism genus, the main point of which is that the main substrate must be able to support growth and metabolism of the micro-organism. When the growth has occurred in an appropriate period of time, on the order of 1 to 7 days, depending on the growth rate of the particular microorganism, a sample of the fermentation broth is analyzed on SPS-ase according to the enzymatic SPS-ase assay as stated in the description.

In order to obtain a more sensitive method for the determination of the enzymatic activity, the temperature can be lowered to 40 ° C and the incubation time can be increased to 20 hours during the determination of the SPS-ase activity, whereby antibiotics must be added to the substrate in order to avoid infection.

By following this test method, other SPS-ase generating 40 microorganisms can be found, both belonging to the genus 8204925 18

Aspergillus as to other genera.

SECTION 5

CHARACTERISTICS OF SOME SPS ASE FORMING MICROORGANISMS

According to the comparative study indicated here for SPS-ase 5-producing microorganisms, it was found that the microorganisms listed in the top part of the following table are SPS-ase producers. The table also contains a strain belonging to the species Asp. japonicus, which is not an SPS-ase producer.

10 ___ _______ SPS-ase Our iden- Official first producer__Type of identification- identifi- depone- yes no Asp. Asp. indication year of the year japo acule designation 15 nicus atus x x A 805 CBS 101.43; DSM 2344 1943 x x A 1443 IFO 4408; DSM 2346 1950 20 x x A 1384 ATCC 20236; DSM 2345 1969 1111 I_L_

A brief identification of the strains indicated above can be found in the following breeding catalogs: 25 List of Cultures 1978 Central Agency for Fungal Cultures, Baarn,

The Netherlands.

Institute for Fermentation Osaka, List of Cultures, 1972, 5th edition, 17-85 Juso-honmachi 2-chome, Yodogawa-ku, Osaka 532, Japan.

The American Type Culture Collection Catalog of Strains I, 14th 30 edition 1980, 12301 Parklawn Drive, Rockville, Maryland 20852.

All strains in the above table closely correspond to the taxonomic description of the Asp species. japonicus and Asp. aculeatus, which appear in The genus Aspergillus of Raper and Fennell, 1965 (see, for example, pages 327-330).

35 SECTION 6 GENERAL DESCRIPTION OF THE COATING TECHNIQUE RELATED TO THE IMMUN0-ELEKTR0F0RESE.

A method referred to as the top agar coating technique has been developed by applicants for identification of individual components of an enzyme complex by cross-immunoelectrophoresis with an 8204925 * 19 polyspecific antibody to all enzyme components in the enzyme complex. The method is based on the fact that enzymes are still active after the specific enzyme-antibody binding or, in other words, that the active enzyme site is not Identical to the enzyme-antibody binding site. The enzyme-antibody complexes deposit as separate arcs in the gel during electrophoresis. The gel plate is covered with soluble SPS in a top agar. After heating at 45 ° C for 20 hours in an atmosphere with a relative humidity of 100%, the arc, which has SPS-ase activity, will appear as a brightening zone in the SPS-cover after precipitation with a mixture of equal parts by volume of ethanol and acetone when looking at it against a black background. Arcs that do not have SPS-ase activity remain invisible.

SECTION 7 IMMUNO ELECTROPHORETIC CHARACTERISTICS OF SPS ASE WITH POLY SPECIFIC ANTI-15 BODY AND COATING.

Rabbits were immunized with the SPSase containing enzyme complex obtained by fermentation of Aspergillus aculeatus CBS 101.43 as indicated in Example I (KEF 68) and the polyspecific antibody was recovered in known manner. By means of this polyspecific antibody, a cross-linked immunoelectrophoresis of the enzyme complex was obtained by fermentation of Asp. aculeatus CBS 101.43, as indicated in Example I, KRF 68, performed as described in NH Axelsen et al., "* A Manual of Quantitative Immunoelectrophoresis", 6 "printing 1977. Reference is made to FIG. 11, which shows the bo-25 gene. that correspond to different proteins produced by the microorganism By means of the above-described top-agar coating technique, it was found that the shaded region corresponds to SPSase.

When the aforementioned hypothesis, which assumes that SPSase consists of at least two enzymes, is correct, the shaded region is the region in which all enzymes responsible for the SPSase activity are present. In other embodiments of the invention, if these enzymes were to be separated by immunoelectrophoresis in such a way that they do not cover each reciprocal region, some of the SPSase activity can be identified by immunoelectrolysis with a coating with both SPS and a commercial spectinase.

SECTION 8

PURIFICATION OF A SPS-ASE PREPARATION

The purification of the SPS-ase preparation KRF 92 (see example I) 8204925 was performed by ion exchange. The buffer is 50nnol Tris (tris-hydroxymethylaminoethane) adjusted to pH 7 with HCl. The column is K 5/30 from Pharmacia, Sweden. The ion exchange material is DEAE-trisacryl from LKB, Bromma, Sweden (300 ml). The flow rate 5 is 100 ml / hour.

15 g of the SPS-ase preparation KRF 92 were dissolved in 450 ml of water at 6 ° C and all of the following indicated operations were performed between 6 ° C and 10 ° C. The pH was adjusted to 7.0 with 1 M Tris. The column was equilibrated with the buffer and then the SPS-ase 10 sample was loaded onto the column. OD 1 and conductivity were measured at the eluate, referring to Figure 12. Fraction 1 is the eluate which is not bound to the ion exchange material. The column is then washed with 2000 ml of buffer to obtain fraction 2. A 0-500 mmol NaCl gradient is now established to obtain the fractions 3-9. All nine fractions were concentrated to 200 ml and dialyzed against water to a conductivity of 2 M mSi by dialysis (Hollow Fiber DP 2 from Amicon, Massachusetts, U.S.A.). The nine fractions were then subjected to a freeze-drying treatment. Only the 20 fractions 1 and 2 showed SPS-ase activity.

Fraction 1 was further purified by gel filtration. 1.5 g of fraction 1 were dissolved in 10 ml of 50 mmol of sodium acetate with a pH of 4.5 (500 mmol of KCl). The column is 2.5 x 100 cm from LKB. The gel filtration filler material is Sephacryl S-200 from Pharmacia, Sweden. The flow rate is 300 ml / hour. The fractions, containing materials with molecular weights between 70,000 and 100,000, calibrated with spherical proteins, contain a factor G designated as an enzyme complex, which SPS cannot decompose when tested according to the qualitative agar test; however, SPS is decomposed according to the qualitative agar test when factor G30 is mixed with a pectinase. It has been found that factor G is able to cleave galactose, fucose and some galacturonic acid from SPS, but the major decomposition product according to the HPLC analysis is still a high molecular weight product very similar to SPS.

SECTION 9 35 pH ACTIVITY DEPENDENCE, TEMPERATURE ACTIVITY DEPENDENCE AND STABILITY OF A SPS ASE.

Fig. 13 shows the pH activity dependence of the SPS-ase preparation KRF 68. From form 2.7 to pH 3.5, a formic acid buffer system was used and from pH 3.7 to 5.5, an acetate buffer system 40 was used.

8204925 κ 21

Fig. 14 shows the temperature activity dependence of the SPSase preparation KRF 68.

Fig. 15 shows the temperature stability of the SPS-ase preparation KRF 68.

5 SECTION 10 ENZYMATIC ACTIVITY DETERMINATIONS.

The table below is a summary of the various enzymatic activity assays pertaining to the invention.

10

Definition of activity unit and description of the enzymatic activity determination type of activity, publicly available later in the short designation description

Enzyme described reference SPS-ase SPS-ase__x __________ 20 Remanence SRU x 1 soluble- SRUM-120 x making____________________________________________

Protease HUT__________x

Cellulase Cr x__ 2 25 __Pu___x____3 PGE__x____4

Pectinase UPTE__x_____5 __PEE__x_____6

Hemi-Icellulase VHCU__x_ 7

The references indicated in the last column of the previous table are detailed in the following table.

8204925 22

Reference can be obtained from {_ (_ NOVO INDUS- Schwei- 5 Refe- IRI A / S, zerische ren- Novo Alle, Ferment AG, a tie Identification of the 2880 Bagsvaerd, Basel, library no. Reference Denmark Switzerland teacup 10 1 Analytical Method AF 154/4 or 1981-12-01 x 2 Analytical Biochemistry 84, 522-532 (1978) ____ x 15 Analytical method AF 149/6-GB or 1981-05-25 x 3 Determination of Pectinase 20 Activity with Citrus Pectin ( PU) or 23.3.1976 x 4 Viscosimetric Poly-galacturonase-Bestimmung 25 (PGE) or 10.11.77 x 5 Bestimmung der Pectin transeliminase (UPTE / g) or 24 Sept. 1975 x 30_____ 6 Determination of the Pectinesterase activity (undated ) with initials WJA / GW x 35_____ 7 Analytical method AF 156/1-GB x 40 In connection with the cellulase activity determination it can be noted that the analysis was performed as indicated in AF 149/6-GB and that the Principles of the assay are explained in Analytical Biochemistry.

SECTION 10a 5 ENZYMATIC DETERMINATION OF SPS ASE.

The enzymatic determination of SPSase is performed in two steps, i.e. a qualitative agar plate assay and a quantitative SPSase activity assay based on the measurement of the amount of total released sugars. When the qualitative agar plate test is negative-10, the SPS-ase activity is zero, without regard to the value derived from the quantitative SPS-ase activity counting assay. When the qualitative agar plate test is positive, the SPS-ase activity is equal to the value obtained from the quantitative SPS-ase activity determination.

15 I. Qualitative agar plate test.

An SPS agar plate was prepared in the following manner. A buffer (B) is prepared by adjusting 0.3 M acetic acid to 4.5 with 1 N NaOH. 1 g of SPS is dissolved in 20 ml of B · 1 g of agarose (HSB Litex) is mixed with 80 ml of B and heated to boiling point with stirring. When the agarose is dissolved, the SPS solution is added slowly. The resulting 125 SPS agarose solution is placed in a water bath at 60 ° C. The plates are now cast by pouring 15 ml of the 125 SFS agarose solution onto a horizontal glass plate measuring 10 cm x 10 cm. Then, 25 places with a distance of 2.5 cm are punched out in the solidified layer of SPS agarose. 10 µl of a 125 solution of the enzyme protein to be tested for SPSase activity is placed in each site. The plate is incubated at 50 ° C and a relative humidity of 100Z for 18 hours. SPS not yet decomposed is precipitated with a solution of equal parts by volume of ethanol and acetone. The SPS-ase agar plate test is positive for a sample placed in a specific location when a transparent annular zone appears around this location.

II. Quantitative SPS-ase activity determination test.

The purpose of this test is to determine enzymatic actives capable of decomposing SPS to such an extent that the decomposition products exhibit greatly reduced or no adsorption or binding affinity for soy protein. Experiments have shown that that portion of the SPS decomposition products, which are not precipitated by a mixture of equal volumes of water and ethanol, do not have any adsorption-binding activity for soy protein.

8204925 24

The SPS-ase assay is based on a hydrolysis of SPS under standard conditions followed by a precipitation of that portion of SPS that is not hydrolyzed with ethanol. After precipitation, the non-precipitated carbohydrate content is determined by quantitative analysis on total sugar (according to AF 169/1, available from NOVO INDUSTRI A / S, 2880 Bagsvaerd).

The standard conditions are: temperature: 50 ° C pH: 4.5 reaction time: control 210 minutes with substrate only, followed by 2 minutes with added enzyme reaction time: main value 212 minutes.

The equipment includes; a shaking water bath with a thermostat at 50 ° C controlled 15 a Whirlimixer mixer centrifuge ice water bath The reagents include: buffer: 0.6 M acetic acid in demineralized water (a) 20 1.0 M NaOH (b) substrate: the pH value of 50 ml of a is adjusted to 4.5 with b, then 4.0 g of SPS are added and after dissolution of the SPS, the pH is adjusted again to 4.5 and the volume is adjusted to 100 ml with deionized water, stop reagent: absolute ethanol.

A SPS-ase activity unit SAE or SPSU is defined as the SPS-ase activity, which releases an amount of 50% ethanol soluble carbohydrate equivalent to 1 pmole galactose per minute under the standard conditions indicated above.

Even if the standard enzyme curve is a straight line in the initial section, it should be noted that it does not intersect point (0.0).

SECTION 10 b 35 ENZYMATIC DETERMINATION OF REMANIENCY SOLUBILIZING ACTIVITY EXPRESSED AS SRUM 120.

Principle

In the method of determining hydrolysis activity, the insoluble portion of degreased, deproteinized and cap-free soybean meal is hydrolyzed under standard conditions. The enzyme reaction 8204925, 4 <25 is stopped with stop reagent and the insoluble part is filtered off. The amount of dissolved polysaccharides is determined spectrophotometrically after acid hydrolysis according to AF 169/1, available from NOVO INDUSTRI A / S, 2880 Bagsvaerd.

Carbohydrases with both endo and exo activity are determined by the method.

The substrate related to this enzymatic assay is identical to the remanence substrate described for the SRU method.

The substrate is dissolved as a 10% solution in the citrate buffer indicated below: 0.1 N citrate-phosphate buffer pH 4.5 5.24 g citric acid 1-hydrate (Merck type 244) 8.12 g disodium hydrogen phosphate 2-hydrate (Merck type 6580) to 1 1 demineralized H2O 15 pH 4.5 + 0.05 stable for 14 days

The stop reagent has the following composition: 100 ml 0.5 N NaOH 200 ml 96% ethanol 20 Store in a refrigerator until use.

Standard temperature conditions 50eC

pH 4.5 reaction time, sample 120 min.

25 reaction time, blank 5 million.

Definition of the unit

A soybean solubilizing unit (SRUM) 120 (M for hand) is the amount of enzyme which liberates solubilized polysaccharides equivalent to 1 ml-30 cromol galactose per minute under the given reaction conditions.

SECTION 10 c ENZYMATIC DETERMINATION OF PR0TE0LYTIC ACTIVITY.

HUT MEASUREMENT

Method for the determination of proteinase in an acidic environment.

The method is based on the digestion of denatured hemo globin by the enzyme at 40 ° C, pH 3.2 for 30 minutes. The digested hemoglobin is precipitated with 14% w / v trichloroacetic acid.

All enzyme samples are prepared by dissolving in 0.1 M acetate-40 buffer at a pH of 3.2.

8204925 26

The hemoglobin substrate is prepared using 5.0 g of lyophilized bovine hemoglobin powder, preserved with 1% thiomer salate and 100 ml of demineralized water, which is stirred for 10 min, then adjusted to 1.7 with 0.33 N HCl. .

After stirring for an additional 10 min., The pH is adjusted to 3.2 with 1 N NaOH. The volume of this solution is increased to 200 ml with 0.2 M acetate buffer. This hemoglobin substrate should be kept in a refrigerator for 5 days.

The hemoglobin substrate is brought to room temperature. At time zero, 5 ml of substrate are added to a test tube containing 1 ml of enzyme. After shaking for 1 second, the tube is placed in a water bath at 40 ° C for 30 min. After exactly 30 min, 5 ml of 14% trichloroacetic acid are added to the reaction tube, which is then shaken and brought to room temperature for 40 min. For the blank test, the hemoglobin substrate is brought to room temperature. At time zero, 5 ml of the substrate are added to a test tube containing 1 ml of enzyme. After shaking for 1 second, the tube is placed in a water bath at 40 ° C for 5 min. After exactly 5 min, 5 ml of 14% trichloroacetic acid are added to the reaction tube 20, which is then shaken and brought to room temperature for 40 min.

After 40 min., The blank and the samples are shaken, filtered once or twice through Berzelius filter No. 0 and placed in a spectrophotometer. The sample is read against the blank test at 275nm while the spectrophotometer is adjusted against water.

Since the absorbance of tyrosine at 275 nm is a known factor, it is not necessary to make a standard curve for tyrosine unless it is necessary to check the Beckman spectrophotometer.

Calculations 1 HUT is the amount of enzyme which forms a hydrolyzate in absorbance at 275 nm in 1 min equivalent to a solution of 1.10 micrograms / ml tyrosine in 0.006 N HCl. This absorbance value is 0.0084. The reaction should take place at 40 ° C, a pH of 3.2 and 35 in 30 minutes.

8204925 5 27 sample blank vol. in ml HUT-x - 0.0084 reaction time in min.

mT_ sample-blank 11 / e „v ,, c HUT - _ x _ (SB) x 43.65 0.0084 30 10 µm / (SB) x 43.65 HUT / g enzyme - * g enzyme in 1 ml

An investigation on the pH stability dependence of the protease in KRF 68 conducted by the HUT analysis with pH values from 2.0 to 8.0 showed that the stability of the protease above pH 8 , 0 was very small, see fig. 16.

In order to illustrate the invention, reference is made to the following Examples I to X, wherein Example I illustrates the preparation of SPSase 20 and Examples II to X illustrate the use of SPSase.

Various fermentations with the Asp. Strains indicated here. aculeatus and Asp. japonicus were performed on a laboratory scale. Hereby, preparations containing SPS-ase according to the SPS-ase test indicated here were obtained. However, since fairly large amounts of SPS-ase are required to perform application trials, similar fermentations were performed on a semi-technical scale, see the following Example I.

Example I

30 Preparation of an SPS axis on a semi-technical scale.

An SPS ase was prepared by immersion fermentation of Aspergillus aculeatus CBS 101.43.

An agar substrate of the following composition was prepared in a Fernbach flask: 8204925 28

Pepton Difco 6 g

Aminoline Ortana 4 g

Glucose 1 g 5 Yeast extract Difco 3 g

Meat extract Difco 1.5 g KH2PO4 Merck 20 g

Malt extract Evers 20 g

H2O 1000 ml treated with ion exchanger 10

The pH was adjusted between 5.30 and 5.35. Then 40 g of Agar Difco were added and the mixture was autoclaved at 120 ° C for 20 min (the substrate is called E-agar).

The CBS 101.43 strain was grown on an inclined E-agar 15 (37 ° C). The traces of the skewed agar were suspended in sterilized skim milk and the suspension lyophilized in vials. The contents of a lyophilized vial were transferred to the Fernbach flask. The flask was then incubated at 30 ° C for 13 days.

A substrate of the following composition was prepared in a seed fermentation machine:

CaCO3 1.2 kg

Glucose 7.2 kg 25 Rofec (dry stopper of corn steep liquor) 3.6 kg soybean oil 1.2 kg

Tap water was added to a total volume of about 240 L. The pH was adjusted to about 5.5 before CaCO3 was added. The substrate was sterilized in the seed fermenter at 121 ° C for 1 hour. The total volume for the inoculation was about 300 l.

The spore suspension from the Fernbach flask was transferred to the seed germination apparatus. Germination fermentation conditions were: 8204925 • * *.

29

Type of conventional aerated and stirred fermenter: fermenter with a height / diameter ratio of about 2.3. Stirring: 300 rpm. (two turbine blades)

Aeration: 300 normal liters of air per minute

Temperature: 30-31®C

Pressure: 50 kPa

Time: about 28 hours 10

About 28 hours after the inoculation, 150 liters were transferred from the seed fermentor to the main fermenter.

A substrate of the following composition was prepared in a 2500 L main fermenter: 15

Heated soy flour 90 kg ΚΗ2Ρ04 20 kg

Pluronic® 150 ml of tap water was added to a total volume of about 900 l. The heated soybean meal was suspended in water. The pH was adjusted to 8.0 with NaOH and the temperature was raised to 50 ° C.

About 925 Anson units of ALCALASE ® 0.6 L were then added to the suspension. The mixture was kept at 50 ° C and pH 8.0 for 4 hours (addition of Na 2 CO 3) without aeration, without pressure and at 100 rpm. stir. Then the remaining substrate components were added and the pH was adjusted to about 6.0 with phosphoric acid. The substrate was sterilized in the main fermenter for 90 min at 123 ° C. The final volume for the inoculation was approximately 1080 30 1.

Then 150 l of seed culture were added.

The fermentation conditions were: 8204925-30

Type of conventional aerated and stirred fermenter: fermenter with a height / diameter ratio of about 2.7. Stirring 250 rpm. (two turbine blades)

Aeration: 1200 normal liters of air per minute

Temperature: 30 ° C

Pressure: 50 kPa

Time ί approximately 151 hours 10

From 24 fermentation hours to about 116 fermentation hours, a pectin solution was aseptically added to the main fermenter at a constant rate of about 8 L per hour. The pectin solution of the following composition was prepared in a 15 500 L dosing tank:

Pectin genu * 22 kg

Conc. phosphoric acid 6 kg

Pluronic® 50 ml 20 * Genu pectin (citrus type NF from The Copenhagen pectin factory Ltd.)

Tap water was added to a total volume of about 325 L. The substrate was sterilized in the dosing tank for 1 hour at 121 ° C. The final volume before the start of dosing was about 360 l. When this portion was discarded, another similar portion was prepared. The total volume of the pectin solution for a fermentation was about 725 L.

After about 151 hours of fermentation, the fermentation process was stopped. The about 1850 L of broth was cooled to about 5 ° C and the enzymes were recovered by the following method.

The culture fluid was filtered through a vacuum drum filter (Dorr Oliver), which was pre-coated with Hy-flo super-cell diatomaceous earth (filtration aid). The filtrate was concentrated to about 15% of the volume of the broth by evaporation. The concentrate was filtered through a Seitz filter sheet (type supra 100) using 0.25% Hy-flo super cell as a filtration aid (referred to as filtration I in the following table). The filtrate was precipitated with 561 g (1 ^ 4) 2804/1 at a pH of 5.5 and 4% Hy-flo super cell diatoms-40 soil is added as a filtration aid. The precipitate and the filtration aid 8204925 31 are separated by filtration through a frame filter. The filter cake is dissolved in water and insoluble portions are separated by filtration over a frame filter. The filtrate is filtered for control over a Seitz filter sheet (type supra 5 100) using 0.25% Hy-flo super cell as a filtration aid (cited in the following table as filtration II). The filtrate is diafiltrated over an ultrafiltration device. After the diafiltration, the liquid is concentrated to a dry matter content of 12.7% (cited in the following table as dry matter content in concentrate).

Optional base treatment for partial removal of the protease activity can be performed at this step. In case the base treatment is carried out, it is carried out at a pH of 9.2 for 1 hour, after which the pH value is adjusted to 5.0.

Now the liquid is filtered for control and filtered for germ reduction purposes and the filtrate is freeze-treated on a freeze-drying equipment from Stokes.

Four fermentations were carried out in the manner described below, varying the strain used for the fermentation, using the optional base treatment and other parameters, as indicated in the following table.

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In order to further reduce the protease activity, a portion of the aforementioned preparations were treated as indicated below using only one of the three alternatives A, B and C.

5 A. 100 g of SPS-ase preparation are dissolved in 1 l of deionized water with stirring at 10 ° C + 2eC. The pH is adjusted to 9.1 with 4 N NaOH. This base treatment is carried out for 1 hour. The pH is then adjusted to 4.5 with glacial acetic acid and dialyzed against ice-cold deionized water to a conductivity of 3 mSi. Freezing treatment and lyophilization are then carried out.

B. 500 g of SPS-ase preparation are dissolved in 4 l of deionized water with stirring at 10 ° C + 2 ° C. The pH is adjusted to 9.1 with 4 N NaOH. This base treatment is carried out for 1 hour. The pH value is adjusted to 5.1 with glacial acetic acid. The obtained material is lyophilized.

C. 50 g of SPS-ase preparation are dissolved in 400 ml of deionized water with stirring at 10 ° C + 2 ° C. The pH is adjusted to 9.1 with 4N NaOH. This base treatment is carried out for 1 hour.

Then the pH is reduced to 5.7 with glacial acetic acid. The obtained material is lyophilized.

SPS-ase preparation used used as starting treatment for code preparation treatment

base ABC

30 KRF 68 x KRF 68 Bil

KRF 68 x KRF 68 EXT

KRF 92 x KRF 92 BI

35 __ (_ | __

The aforementioned preparations are characterized by their activities of the enzymes with respect to the invention in the following table.

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Example II (application example)

This example describes the production of a p.v.p. soybean meal "Sojamei 13", liberated and degreased from caps, (commercially available from Aarhus Oliefabrik A / S). The dry matter content of this flour 5 was 94.0% and the (N x 6.25) dry matter content was 58.7%. The soybean meal was treated with the SPS-ase preparations KRF 68 B II (Example I) in the following manner: 85.2 g of the soybean meal were suspended and stirred at 50 ° C into 664.8 g of water and the pH was adjusted by means of of 7.5 ml of 6 N HCl at 4.5 in-10. 50 g of a solution containing 4.00 g of the SPS-ase preparation were added and the reaction mixture was then stirred at 50 ° C for 240 ml. The mixture was then centrifuged in a laboratory centrifuge (Beekman Model J-6B) for 15 ml at 3000 x g. The supernatant was weighed and analyzed for 15 Kjeldahl nitrogen and dry matter. Then the solid base was washed with a volume of water equivalent to the mass of the supernatant obtained from the first centrifugation. This treatment was performed twice. The solid base was then lyophilized, weighed and analyzed for Kjeldahl N and dry matter at Qvist's Laboratory, Marselis Boulevard 169, 8000 Aarhus C, Denmark. This laboratory is authorized by the state for analyzes on feed and milk products. The results obtained in this experiment are shown in Table 2.1.

8204925 36

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Therefore, a p.v.p. just obtained a protein purity, i.e., N x 6.25 on a dry matter basis, of 91.4% and with a total protein yield of 83Z.

Example III (Application Example) This example was performed to compare the protein yields, food quality and some functional properties of soy protein products prepared by the following three methods: A: The traditional isoelectric precipitation for the preparation of isolated soy protein.

10 B: The traditional isoelectric washing treatment for the preparation of concentrated soy protein.

C: The isoelectric washing treatment of the invention including a remanence solubilizing enzyme for the preparation of p.v.p.

In order to develop a true comparison of the method of the invention (C) with the conventional soy protein processes (A and B), the same raw material has been used in all cases. Also, the experiments were conducted in such a way that corresponding temperatures and treatment times are the same in all three cases.

Only the pH values were different due to the fundamental differences between the three experiments.

A. The traditional isoelectric precipitation for the preparation of isolated soy protein.

425.8 soybean meal (Soyamel 13 prepared by Aarhus Oliefabrik A / S) was extracted in 3574.2 g of tap water at 50 ° C. The pH was adjusted to 8.0 with 20.1 g of 4 N NaOH. After stirring for 1 hour, the suspension was centrifuged at 3000 x g for 15 min using 4 1 L beakers in a laboratory centrifuge (Beckman Model J-6B). The centrifuge product I and the precipitate I were weighed. The precipitate I was extracted again with water to a total weight of 4000 g. The temperature was kept at 50 ° C, the pH was adjusted to 8 with 4 N NaOH and the suspension was stirred for 1 hour. Centrifugation and weighing of centrifuge product II and precipitate II were performed as before. Samples were taken from the centrifuge products I and II and the precipitate II for Kjeldahl and dry matter determinations. The centrifuge products I and II were then mixed and kept at 50 ° C. The protein was then isoelectrically precipitated at pH

4.5 by means of 45 g of 6 N HCl. After stirring at 50 ° C for 1 hour, the protein was collected by centrifugation at 3000 x g for 15 min.

The centrifuge product III was weighed and analyzed for Kjeldahl N

8204925 38 and dry matter. The solid phase III was weighed and washed with water in an amount corresponding to the weight of centrifuge product I. The washing treatment was carried out by stirring at 50 ° C for 1 hour. The washed protein was collected by centrifugation at 3000 x g for 15 min. The centrifuge product IV and the solid phase IV were weighed. Centrifuge product IV was analyzed for Kjeldahl N and dry matter. The solid phase was suspended in 1550 g of water at 50 ° C and the pH was adjusted to 6.5 with 17 g of 4 N NaOH. The mixture was stirred for 1 hour and adjusted to pH 6.5 again if necessary.

Finally, the product was subjected to a freeze-drying treatment, weighed and analyzed for Kjeldahl N and dry matter. The mass balance calculations are listed in Table 3.1.

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8204925 41 B. The Isoelectric Washing Treatment for the Preparation of Soy Protein Concentrate._ ** • 111111 *** ™ ·········· * ················· · ™ ······ ™ ······· ^^ 425.6 g of soybean meal (Soybean egg 13 prepared by Aarhus Oliefabrik A / S) were washed in 3574 g of water at 50 ° C. The pH was adjusted to 4.5 with 44.8 g of 6 N 5 HCl. The wash was performed by stirring for 4 hours. The suspension was then centrifuged at 3000 x g for 15 min using 4 1 L beakers in a laboratory centrifuge (Beckman Model J-6B). The centrifuge product I was weighed and analyzed for Kjeldahl N and dry matter. The solid phase I 10 was weighed and washed again with water to a total weight of 4000 g. The pH was reset to 4.5 with 1.7 g of 6 N HCl and the suspension was stirred at 50 ° C for 30 min. Centrifugation and weighing of centrifuge product II and solids II was performed as before. The solid phase II was resuspended in 1575 g of water at 50 ° C and the pH was adjusted to 6.5 with 34.5 g of 4 N NaOH. The mixture was stirred at 50 ° C for 1 hour and adjusted to pH 6.5 if necessary. Finally, the protein product was subjected to a freeze drying treatment, weighed and analyzed for Kjeldahl N and dry matter. The mass balance is shown in table 3.2.

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425.8 g of soybean meal (Soybean Egg 13 prepared by Aarhus Oliefabrik A / S) was washed in 3524.2 g of water at 50 ° C. The pH was adjusted to 4.5 using 43.7 g of 6 N HCl. 24 g of the SPS-ase preparation KRF 68 5 III (Example I) were dissolved in 26 g of water and added to the wash mixture. The wash was then carried out with stirring for 4 hours. Purification was then carried out as described for B, with the amounts of 6 N HCl, 4 N NaOH and water before resuspending being the only parameters with different values. The mass balance is given in table 3.3.

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8204925 47

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The amino acid compositions of the three protein products were determined, see Table 3.4. Total essential amino acid content, chemical state and essential amino acid index (EAAI) are calculated using the FAO reference pattern from 1957.

The trypsin inhibitor content of the three products was determined by the method described in A.O.C.S. Tentative Method Ba 12-75 (A.O.C.S. is short for American Oil Chemists' Society). The results are included in Table 3.5, which also contains the yields and the protein / dry matter ratio of the three products.

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Functional properties

The nitrogen soluble heroin index (NSI) was determined in a 125 protein protein at pH 7.0 in 0.2 M NaCl, respectively. In distilled water. After stirring for 45 ml with a magnetic stirrer, the suspension 5 was centrifuged at 4000 x g for 30 ml and the supernatant was analyzed for nitrogen. The nitrogen solubility was calculated as (soluble NZ / total NZ). The results of this valuation for the three products are shown in Table 3.6.

The emulsifying power was determined three times for each product according to a slightly modified Swift titration. 4.0 g (N x 6.25) of the product were mixed in 250 ml 0.5 M NaCl with a Sorval Omni mixer at low speed. 50 ml of the suspension was transferred to a glass mixing cup and 40 ml of soybean oil was added. After this, the total mixture was weighed · The oil-water mixture was then mixed at 10,000 rpm. homogenized with the beaker in an ice bath. An additional amount of soybean oil was added at a rate of 0.3 ml per sec. until the emulsion collapsed. The total amount of added oil for the "endpoint" was found by weighing.

The emulsifying power was calculated as ml oil per g protein 20 (N x 6.25). The density of the oil was taken as 0.5 g / ml.

The average results of the determination of the emulsifying power of the three products are shown in Table 3.6.

The beaten expansion was determined in a 3Z's protein solution at pH 6.5. 250 ml of the aqueous dispersion of the protein samples 25 were beaten at speed III for 4 min in a Hobart mixer (model N-50) equipped with a wire whipper. The beaten expansion was calculated according to the formula V-250 beaten expansion ..... 1111 x 100Z, 30 where V = * the beaten final volume in ml.

V was measured by refilling the mixing cup with water.

Duplicates were run for each of the three samples. The average results are shown in Table 3.6.

The foam stability was determined as the ratio between the amount of foam remaining after 30 ml spouts and the original amount of foam. 1 g of foam obtained by the previous method was placed in a plastic cylinder (diameter 7 cm, height 9 cm) with a wire net with a mesh width of 1 mm x 1 mm. The cylinder 40 was placed on a funnel on top of a glass cylinder and the weight (B) of spilled liquid in the glass cylinder was determined. The foam stability FS is defined by the equation FS = x 100%

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The results of the determination are shown in Table 3.6.

The gel strength is defined in this specification as the Brookfield viscosity measured by T-axes on a Brookfield Helipath tripod. The gels were prepared by heat treatment of 12% protein suspensions in 0.5 M NaCl. The heat treatment was carried out in closed jugs with a diameter of 7.3 cm and a height of 5.0 cm placed in a water bath, maintained at 80 ° C and 100 ° C for 30 minutes each. The jugs were cooled and thermostat-controlled at 20 ° C before opening and measuring. The results of the measurements are shown in Table 3.6.

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Example IV (application example)

A p.v.p. was prepared according to the method described in Example III C, except that the cellulase activity was partially derived from Trichoderma reseei. The commercially available cellulase preparation 5 CELLUCLAST prepared by Novo Industrie A / S was treated with a low temperature base in the following manner. The pH of a 10% aqueous CELLUCLAST solution was adjusted to 9.2 with NaOH and the resulting solution was cooled to 5 ° C. After 1 hour at this pH and this temperature, the pH was reset to 4.7 with 20% acetic acid. This solution was kept at 5 ° C overnight and then sterile filtered. The filtrate was subjected to a freeze-drying treatment. 4 g of the product obtained by freeze-drying were added together with the SPS-ase preparation KRF 68 B III (example I). The two enzymes were solubilized in 172 g of water before addition to the wash mixture. The mass balance determinations of this example are given in Table 4.1.

The experiment shows that this particular SPSase preparation already contains an effective cellulase since addition of CELLUCLAST does not seem to affect the protein / dry matter ratio. However, other SPSase preparations may contain less cellulase, for example KRF 92, see the table immediately preceding Example II.

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Example V (application example)

According to the method described in Example III C, a p.v.p. except that all weights were reduced by a factor of 5 and the reaction mixture was cooled to about 5 ° C prior to centrifugation. On the basis of the analysis results in connection with the centrifuge products, a theoretical yield of precipitated protein is obtained, as stated in table 5.1.

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8204925 59

Example VI (example of application)

Demonstration of the protein content of SPS

40 g (N x 6.25) of an isolated commercial soy protein product (Purina 500 E from Ralston Purina) were dissolved in 680 g of water.

The mixture was heated to 50 ° C in a water bath and the pH was adjusted to 4.50 with 6N HCl. 90 g of this mixture were transferred to five 250 ml Erlenmeyer flasks and 10 g of aqueous solutions, respectively. 0g, 0.2g, 0.4g, 0.8g and 1.6g of the SPS, prepared as described earlier in the description, were added. The flasks were then stirred with a magnet in a water bath at 50 ° C for 240 min.

After this, the suspensions were centrifuged at 3000 x g for 15 min and the centrifuge products I were analyzed for Kjeldahl N and dry matter. The solid phases were washed in water at room temperature and recentrifuged. This method was repeated. The solids were then dispersed in 50 ml of water and the pH was adjusted to 6.50 by the dropwise addition of 6 N NaOH. The neutralized products were subjected to a freeze-drying treatment and analyzed for Kjeldahl N and dry matter. Based on the analyzes listed in Table 6.1, the protein recovery and the percentage of SPS bound to the protein are calculated using the formulas listed in connection with Table 6.2.

This example shows that the SPS is tightly bound to the protein, so that the protein / dry matter ratio decreases with increasing SPS content. An SPS content comparable to about 0.4 g in 10 g of water added to 50 g of isolated protein product is the protein / SPS ratio present in the soybean meal.

The X binding of SPS is a calculated value. The% binding of SPS decreases due to saturation of the protein relative to SPS at the low protein / SPS ratios.

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Example VII (application example)

This example describes the preparation of a p.v.p. using the SPS-ase preparation KRF 92 B1 in a dose of 5% of the dry matter. The method of preparation was exactly as in Example III C, 5 except that all weights were reduced by a factor of 5. The p.v.p. was analyzed as described in Example II. The results obtained in the experiment are shown in Table 7.1.

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Example VIII (application example)

This example illustrates the effect of pretreatment of soybean meal by jet cooking prior to the preparation of p.v.p.

Pre-treatment

A suspension of soybean meal in water consisting of 10 kg of soybean meal (Soybean 13 prepared by Aarhus Oliefabrik A / S) per 100 kg was pumped through a steam injector (type Hydroheater B-300) and with steam of 8 bar in such an amount and with a flow mixed such that a final temperature of 150 ° C for 25 sec. could be maintained in a tubular pressurized reactor. After this, the pressure was released in a relaxation room (a cyclone) and from there the suspension was passed through a plate heat exchanger and cooled to about 50 ° C. The cooled suspension could be used directly for the preparation of p.v.p. according to the invention, but in this case the suspension was spray dried at an inlet temperature of 200 ° C and an outlet temperature of 90 ° C. The pretreated product was found to have a dry matter content of 96.5% and a protein content of 56.9% (N x 6.25).

Preparation of p.v.p.

This preparation was carried out in the following manner: 70 g of dry matter of the jet-boiled and dried soybean meal was suspended and stirred in 560 g of water at 50 ° C and the pH was adjusted to 4 by 6.5 ml of 6 N HCl at 4 , 50 set. 6 x 90 g of this suspension were transferred to six 250 ml Erlenmeyer flasks and stirred on a water bath at 50 ° C by magnetic stirrers. 10 g of a solution were added to each flask, which were resp.

0 g, 0.025 g, 0.050 g, 0.10 g, 0.20 g and 0.40 g of the SPSase preparation KRF 68 B III. The reaction mixtures were then stirred at 50 ° C for 240 min. Centrifugation was then performed at 3000 x g for 15 min.

The supernatant was then analyzed for Kjeldahl N and the solid phase was washed with water of equal volumes and centrifuged. This method was performed twice. The solid phase was then subjected to a freeze-drying treatment and analyzed for Kjeldahl M and dry matter.

A similar experiment was performed with an untreated soybean meal (Sojamei 13 from Aarhus Oliefabrik A / S) as starting product. In this case, the enzyme substrate ratios were 0%, 1%, 2%, 3%, 4% and 8%. 40 Based on the protein content of the above layers, the 8204925 * 65% percentage of protein recovered can be calculated. The efficiency of the protein is based on the assumption that the enzyme product is made 100% soluble after the reaction. The table below shows the results obtained by both experiments.

5

Table 8.1 Protein efficiency and protein dry matter ratio for p.v.p. prepared from cooked and untreated soy flour.

_boiled soybean meal untreated soybean meal_ 10 U / S% Protein Protein of Protein Protein of yield% dry matter% yield% dry matter% * 0 92.9 76.5 90.7 73.9 0.25 90.1 86.6 15 0 50 89.3 88.7 1.0 88.1 89.7 87.1 86.2 2.0. 86.6 91.7 85.7 88.1 3.0 - - 84.3 89.5 4.0 84.7 92.2 82.6 90.9 20 8.0 - - 76.2 91.1

Example IX

Isolation of protein from corn gluten.

Corn gluten is usually produced as a by-product in the corn starch manufacturing process. This impure corn gluten is not efficiently separated from the fiber fraction and this is one of the reasons why it is without any valuable functional properties. Also, various degradable polysaccharides impure corn gluten.

A series of SPS-ase treatments were carried out with an SPS-ase preparation obtained according to Example I, except that an ultrafiltration was performed instead of the (1 ^ 4) 2804 precipitation, whereby the isolated enzyme was treated with a base according to the method A 35 (this base-treated SPSase preparation is referred to as PPS 1305 for brevity for the sake of brevity). PPS 1305 contains virtually no proteolytic activity.

The following reaction conditions were used: 8204925 66

Substrate concentration: S 10% dry matter (Staley corn gluten) pH = 4.50

T »50 ° C

5 to 240 minutes

Enzyme: PPS 1305 E / S% «0; 1; 2; 3; 4; 8%

The protein product was purified by centrifugation, washing twice and finally subjected to a freeze-drying treatment.

10 The results are shown in the following table 9.

Table 9 15 Experiment Enzyme Dose Protein Dissolved Poly-Protein Pure No. E / S% efficiency% saccharide% (Nx6.25 / DM)% 873 0 96 0 64 874 1 95 40 75 20 875 2 95 50 78 876 3 95 52 79 877 4 95 63 83

Example X

Isolation of protein from cottonseed meal, sunflower meal and turnip seed meal.

The protein from cottonseed meal, sunflower meal or rapeseed meal can be isolated in the same way as the protein from soybean meal and corn gluten. Isolation of protein from various other protein-containing vegetable raw materials can be performed in the same manner. Of course, the isolation and / or the separation should preferably be carried out at the isoelectric point of the main part of the protein part of the starting product, the protein yield being maximum.

8204925 r 67

An overview of the figures already referred to is given below for the purpose of providing a more concise overview.

Fig. Belongs To Describes 5 No.

1 the general part demonstrating the binding effect of the description between SPS and soy protein 10 2 the general part the flow chart, which describes the preparation of the description SPS 3 section 2 the calibration curve for HPLC gel filtration chromatography 15_____

4 section 2 the HPLC gel filtration chromatogram of SPS

5 section 2 the HPLC gel filtration chromatogram of SPS

decomposed by SPS-ase 20________ 6 sections 2 and 3 the HPLC gel filtration chromatogram of the supernatant from SPS incubated with soy protein 7 section 2 the HPLC gel filtration chromatogram of the supernatant from dissected SPS incubated with soy protein

8 section 3 the HPLC gel filtration chromatogram of APS

Decomposed by pectolyase

9 Section 3 The HPLC Gel Filtration Chromatogram of APS

decomposed by SPS-ase

35 10 section 3 the HPLC gel filtration chromatogram of SPS

treated with pectolyase 11 section 7 the immunoelectrophoretic peaks including an SPS-ase peak identified 40 by a coating technique 8204925 68 (continued) 12 section 8 the ion-exchange chromatogram of an SPS-ase 5___ 13 section 9 the pH activity dependence of an SPS -ase 14 section 9 the temperature activity dependence 10 of an SPS-ase 15 section 9 the temperature stability of an SPS-ase 16 section 10 the pH stability of protease in an SPS-ase preparation. 1__ * 8204925

Claims (14)

1. Agent for the decomposition of vegetable retentivity, In particular soy maneness, in the presence of vegetable protein, In particular soy protein, suitable for the preparation of a pvp with a protein purity of about 90%, with a vegetable protein that is degreased or may be partially degreased, as a starting product, containing an enzyme with remanence solubilizing activity, characterized in that the agent contains an enzyme which is capable of decomposing soybean SPS (SPS-ase) and wherein the agent is essentially ls of proteolytic activity Agent according to claim 1, characterized in that the SPSase is prepared by means of a microorganism belonging to the genus Aspergillus, preferably belonging to the Aspergillus niger group. Agent according to claim 1 or 2, characterized in that the active component comes from the enzymes obtainable by means of Asp. aculeatus CBS 101.43. Agent according to claims 1 to 3, characterized in that the SPS-axis is lramuno electrophoretic identical to the SPS-axis, which can be prepared by means of Asp. aculeatus CBS 101.43 and identifiable by the immunoelectrophoretic coating technique. Agent according to claim 3 or 4, characterized in that the ratio between the proteolytic activity in HUT units and the remanence solubilizing activity in SRUM-120 units is less than about 2: 1, preferably less than 1: 1, more preferably less than 0.25: 1. Agent according to claims 1 to 5, characterized in that the agent contains cellulase activity and that the cellulase activity originates in part or in whole from Trlchoderma reseei. Agent according to claims 1 to 6, characterized in that the agent contains cellulase activity (C *), pectinase activity (PU, PGE, UPTE, PEE) and hemicellulase activity (VHCU). 8. A process for preparing a purified vegetable protein product for removal of remanence from an impure vegetable protein serving as raw material, characterized in that the starting product is treated with the agent according to claims 1 to 7 in an aqueous medium at a pH value which differs by no more than 1.5 pH units from the isoelectric point of the major part of the protein portion of the starting product and at a temperature of between about 20 and about 70 ° C, to at least about 60% of the 8204925 remanence, based on the nitrogen and dry matter mass balance, preferably at least about 70% thereof, more preferably at least about 80% thereof, is solubilized, after which the solid phase purified vegetable protein product, separates from the top layer. Process according to claim 8, characterized in that the separation is carried out at a temperature between room temperature and the freezing point of the above layer. 10. Process according to claim 8 or 9, characterized in that 10 is used as starting product degreased or degreased and furthermore partially purified vegetable protein. Process according to claims 8 to 10, characterized in that soybean meal is used as the starting product. 12. Process according to claims 8 to 11, characterized in that heat-treated soy flour, preferably jet-cooked soy flour, is used as the starting product. Process according to claims 8 to 12, characterized in that an initial product is used which passes through a sieve with a mesh size of approximately 2.5 nm. Purified vegetable protein product obtained by one or more of the method according to claims 8 to 13. 8204925